Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) exists in many different sub- populations within diverse host milieus. In order to target all Mtb populations effectively, TB treatment is lengthy and requires multiple antibiotics. The mechanisms that underlie formation of these phenotypically distinct bacteria are poorly understood and their characterization is needed in order to improve TB therapy. We hypothesize that a distinct form of ribosomes containing alternative ribosomal proteins (AltRPs) allows for the tuning of protein synthesis in the response of Mtb to environmental signals. One of these signals may be zinc availability, which changes from very high concentrations experienced by intracellular Mtb in macrophages to a zinc depleted niche where extracellular Mtb is found. Zinc depletion is likely caused by a large amount of zinc-binding protein calprotectin released from lysed neutrophils in necrotic granulomas, the hallmark of a poorly controlled TB infection. According to our preliminary data, Mtb responds to calprotectin by increasing the number of AltRPs-containing (Alt) ribosomes, which we predict is a means of adapting protein synthesis to the changing host environment. Alt ribosomes may have different specificity and/or activity compared the primary (Prim) ribosomes. Our goals are to identify transcription factor(s) and conditions that drive expression of the altRP operon; to investigate functional effects of replacing PrimRPs with the AltRPs in stress susceptibility; to identify proteins that are preferentially synthesized by the Alt ribosomes and propose mechanisms that allow this selection. We will use a DNA fragment containing altRP promoter to pull-down protein(s) from Mtb cell lysate and genetic tools to identity transcription factors that regulate altRP expression. We will also quantify altRP mRNA in granulomas from infected monkeys, focusing on necrotic granulomas, and cultures exposed to various stresses, which mimic those in necrotic granulomas. Further, to assess the role of the Alt ribosomes in adaptation to a transition from intracellular to extracellular environment in necrotic granulomas, we will test growth and survival of the altRP deletion mutant by combining the macrophage infection model followed by the exposure to calprotectin and other stresses expected in vivo. In addition, we will employ mass spectrometry to identify proteins that are synthesized by Prim vs. Alt ribosomes. Finally, we will investigate potential conserved sequences on mRNAs that may allow selection by Alt ribosomes in order to be able to predict which proteins are expected to be synthesized in neutrophil-rich niche. Deciphering the role of Alt ribosomes will provide insights into a novel mechanism of translation regulation in Mtb that may be relevant to the formation of population diversity and phenotypic drug resistance. Importantly, inhibitors of the Alt ribosomes would be active against bacteria residing in zinc depleted niches such as those existing in necrotizing granulomas and might provide a novel approach to shortening and improving TB treatment.